Diagnosing and treating alzheimers disease

ABSTRACT

Described herein are methods, systems and compositions for the diagnosis, prognosis and treatment of dementia and Alzheimer&#39;s disease. Also described are methods, systems and compositions to distinguish between Alzheimer&#39;s disease and Parkinson&#39;s disease. In various embodiments levels of PACAP and/or SIRT3 are analyzed for the diagnosis, prognosis and treatment of dementia and Alzheimer&#39;s disease.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/276,771, filed Feb. 15, 2019, currently pending, which is acontinuation of U.S. patent application Ser. No. 15/962,691, filed Apr.25, 2018, now U.S. Pat. No. 10,247,738, which is a continuation of U.S.patent application Ser. No. 15/103,059, filed Jun. 9, 2016, now U.S.Pat. No. 9,989,541, which is a National Phase of InternationalApplication No. PCT/US2014/072216, filed Dec. 23, 2014, now expired,which claimed priority under 35 U.S.C. § 119(e) to U.S. ProvisionalPatent Application No. 61/921,359, filed Dec. 27, 2013, now expired, thecontents of which are herein incorporated by reference in theirentirety.

BACKGROUND

All publications herein are incorporated by reference to the same extentas if each individual publication or patent application was specificallyand individually indicated to be incorporated by reference. Thefollowing description includes information that may be useful inunderstanding the present invention. It is not an admission that any ofthe information provided herein is prior art or relevant to thepresently claimed invention, or that any publication specifically orimplicitly referenced is prior art.

Pituitary Adenylate Cyclase Activating Polypeptide (PACAP), as a smallpeptide with either 38 amino acids in full length form (PACAP-38), or 27amino acids in short form (PACAP-27), is broadly recognized as aneurotrophin associated with stress. Both forms strongly increase cyclicadenosine monophosphate (cAMP) by activating adenylate cyclase, andhence named as PACAP. Subsequent research showed that PACAP is not onlyan endocrine hormone, but intrinsically expressed in multiple brainregions and peripheral tissues. PACAP is a potent neurotrophic andneuroprotective peptide in the central nervous system (CNS). PACAP-38 isthe major form in brain, while PACAP-27 exists in minor quantity. Bothforms of PACAP bind to and activate G protein-coupled receptors (PAC1,VPAC1, and VPAC2). PAC1 is mainly localized in the CNS; while VPAC1 andVPAC2 are in the vascular system and the gastrointestinal tract.

PACAP has been shown to promote synaptic transmission, long termpotentiation and memory under physiological conditions. However, therelevance of PACAP expression has not been extensively studied in thehuman brain, including those suffering from Alzheimer's disease (AD), aprogressive mental deterioration and form of dementia that often occursin old age due to generalized degeneration of the brain. AD is aneurodegenerative disorder that affects memory and other cognitivefunctions, and is the most common cause of dementia. Alzheimer's DiseaseAssociation (ADA) survey shows that 5.4 million people in the UnitedStates (US) currently have AD and 13.5 million are expected to have ADwithin the next 40 years. AD affects over 26 million people worldwideand currently there is no cure for the disease. With the growing numberof people living to older ages, there is an urgency to better understandelements of the pathogenic pathway, discover agents that target theseelements, and establish their roles in the treatment and prevention ofAD. But effective biomarkers and treatment are lacking. There is nodisease modifying medication available on the market.

Thus, there is a need in the art for novel and effective treatments andmethods of diagnosing Alzheimer's disease and other forms of dementia.

SUMMARY OF THE INVENTION

The following embodiments and aspects thereof are described andillustrated in conjunction with compositions and methods which are meantto be exemplary and illustrative, not limiting in scope.

Various embodiments of the present invention provide for a method ofdiagnosing dementia and/or a neurological condition in an individual,comprising: obtaining a sample from the individual; preparing the sampleto be assayed by a serological test to determine the presence or absenceof low SIRT3 expression levels and/or low PACAP expression levels; anddiagnosing dementia and/or a neurological condition based on thepresence of low SIRT3 expression levels and/or low PACAP expressionlevels relative to those levels found in a healthy subject.

In various embodiments, the sample to be assayed by a serological testcan be prepared to determine the presence or absence of low PACAPexpression, and dementia and/or a neurological condition can bediagnosed based on the presence of low PACAP expression levels relativeto those levels found in a healthy subject.

In various embodiments, the sample to be assayed by a serological testcan be prepared to determine the presence or absence of low SIRT3expression, and dementia and/or a neurological condition can bediagnosed based on the presence of low SIRT3 expression levels relativeto those levels found in a healthy subject.

In various embodiments, the dementia can be Alzheimer's disease (AD).

Various embodiments provide for a method for determining whether asubject has dementia and/or a neurological condition, comprising:obtaining a biological sample from a subject; determining the PACAPexpression level and/or the SIRT3 expression level in the biologicalsample; and diagnosing the subject as having dementia if low PACAPexpression levels and/or low SIRT3 expression levels relative to a PACAPreference value and/or SSIRT3 reference value is found in the biologicalsample.

In various embodiments, determining the PACAP expression level orassaying the biological sample to determine PACAP expression level cancomprise contacting the biological sample with an anti-PACAP antibodyand detecting whether binding occurs or the amount of binding occurringbetween PACAP protein and the anti-PACAP antibody.

In various embodiments, determining the SIRT3 expression level orassaying the biological sample to determine SIRT3 expression level cancomprise contacting the biological sample with an anti-SIRT3 antibodyand detecting whether binding occurs or the amount of binding occurringbetween SIRT3 protein and the anti-SIRT3 antibody.

Various embodiments provide for a method of distinguishing betweenAlzheimer's disease and Parkinson's disease, comprising: obtaining abiological sample from a subject desiring a distinction betweenAlzheimer's disease and Parkinson's disease; assaying the biologicalsample to determine PACAP expression level; and determining the subjecthas Alzheimer's disease if the PACAP expression level is lower than areference value, or determining the subject does not have Alzheimer'sdisease if the PACAP expression level is not lower than the referencevalue.

In various embodiments, the method can further comprise assaying thebiological sample to determine SIRT3 expression level; and determiningthe subject has Alzheimer's disease if the PACAP expression level andthe SIRT3 expression level are lower than their reference values, ordetermining the subject does not have Alzheimer's disease if the PACAPexpression level and the SIRT3 expression level are not lower than theirreference values.

Various embodiments provide for a method of prognosing dementia and/or aneurological condition in an individual, comprising: obtaining a samplefrom the individual; preparing the sample to be assayed by a serologicaltest to determine the presence or absence of low PACAP expression levelsand/or low SIRT3 expression levels; and prognosing a severe form ofdementia and/or a neurological condition based on the presence of lowPACAP expression levels and/or low SIRT3 expression levels relative tothose levels found in a healthy subject.

In various embodiments, the sample to be assayed by a serological testcan be prepared to determine the presence or absence of low PACAPexpression, and a severe form of dementia and/or a neurologicalcondition can be prognosed based on the presence of low PACAP expressionlevels relative to those levels found in a healthy subject.

In various embodiments, the sample to be assayed by a serological testcan be prepared to determine the presence or absence of low SIRT3expression, and a severe form of dementia and/or a neurologicalcondition can be prognosed based on the presence of low SIRT3 expressionlevels relative to those levels found in a healthy subject.

In various embodiments, the dementia is Alzheimer's disease (AD).

Various embodiments provide for a method of diagnosing susceptibility toAlzheimer's disease, comprising: obtaining a sample from the individual;preparing the sample to be assayed by a serological test to determinethe presence or absence of low SIRT3 and/or PACAP expression levels; anddiagnosing susceptibility to Alzheimer's disease based on the presenceof low SIRT3 and/or PACAP expression levels relative to those levelsfound in a healthy subject.

In various embodiments, the sample to be assayed by a serological testcan be prepared to determine the presence or absence of low SIRT3expression, and the individual can be diagnosed to be susceptible toAlzheimer's disease based on the presence of low SIRT3 expression levelsrelative to those levels found in a healthy subject.

In various embodiments, the sample to be assayed by a serological testcan be prepared to determine the presence or absence of low PACAPexpression, and the individual can be diagnosed to be susceptible toAlzheimer's disease based on the presence of low PACAP expression levelsrelative to those levels found in a healthy subject.

Various embodiments provide for a method of diagnosing susceptibility toa neurological disorder, comprising: obtaining a sample from theindividual; preparing the sample to be assayed by a serological test todetermine the presence or absence of low SIRT3 and/or PACAP expressionlevels; and diagnosing susceptibility to the neurological disorder basedon the presence of low SIRT3 and/or PACAP expression levels relative tothose levels found in a healthy subject.

In various embodiments, the sample to be assayed by a serological testcan be prepared to determine the presence or absence of low PACAPexpression, and the individual can be diagnosed to be susceptible to theneurological disorder based on the presence of low PACAP expressionlevels relative to those levels found in a healthy subject.

In various embodiments, the sample to be assayed by a serological testcan be prepared to determine the presence or absence of low SIRT3expression, and the individual can be diagnosed to be susceptible to theneurological disorder based on the presence of low SIRT3 expressionlevels relative to those levels found in a healthy subject.

A method of treating dementia and/or a neurological condition in anindividual, comprising: providing a composition comprising PACAP, SIRT3,salts thereof, analogs thereof, derivatives thereof, pharmaceuticalequivalents thereof, or combinations thereof; and administering atherapeutically effective dosage of the composition to the individual.

In various embodiments, the composition can be administered to theindividual intravenously.

In various embodiments, the dementia can be Alzheimer's disease (AD).

Various embodiments provide for a method of treating dementia and/or aneurological condition, comprising: providing a composition comprisingone or more agonists of receptors to PACAP, SIRT3, or combinationsthereof, and administering a therapeutically effective dosage of thecomposition to the individual. In various embodiments, the dementia isAlzheimer's disease.

Various embodiments provide for a method of treating dementia and/orneurological condition, comprising: providing a composition comprisingone or more neurotrophins; and administering a therapeutically effectivedosage of the composition to a subject who has been determined to havedementia and/or the neurological condition based on the subject's PACAPlevel and/or SIRT3 level.

Various embodiments provide for a method for treating dementia and/orneurological condition in a subject, comprising: analyzing a biologicalsample from the subject for a PACAP level; and administering aneurotrophin or an agonist of a neurotrophin to the subject when thePACAP level is below a reference value to treat dementia and/orneurological condition.

In various embodiments, the method can further comprise: analyzing thebiological sample for a SIRT3 level and the neurotrophin or the agonistof a neurotrophin can be administered to the subject when the PACAPlevel is below a PACAP reference value and the SIRT3 level is below aSIRT3 reference value to treat dementia and/or neurological condition.

Various embodiments provide for a method for treating dementia and/orneurological condition in a subject, comprising: obtaining the resultsof an analysis of a PACAP level in a subject; and administering aneurotrophin or an agonist of a neurotrophin to the subject when thePACAP level is below a reference value.

In various embodiments, the method can further comprise obtaining theresults of an analysis a SIRT3 level and the neurotrophin or the agonistof the neurotrophin can be administered to the subject when the PACAPlevel is below a PACAP reference value and the SIRT3 level is below aSIRT3 reference value to treat dementia and/or neurological condition.

Various embodiments provide for a method for treating dementia and/orneurological condition in a subject who has been determined to have aPACAP level below a reference value, comprising: administering aneurotrophin or an agonist thereof to the subject. In variousembodiments, the subject has also been determined to have a SIRT3 levelbelow a SIRT3 reference value.

Various embodiments provide for a method for treating dementia and/orneurological condition in a subject, comprising: analyzing a biologicalsample from the subject for a SIRT3 level; and administering a SIRT3 oran agonist thereof to the subject when the SIRT3 level is below areference value to treat dementia and/or neurological condition.

Various embodiments provide for a method for treating dementia and/orneurological condition in a subject, comprising: obtaining the resultsof an analysis of a SIRT3 level in a subject; and administering SIRT3 oran agonist thereof to the subject when the SIRT3 level is below areference value.

Various embodiments provide for a method for treating dementia and/orneurological condition in a subject who has been determined to have aSIRT3 level below a reference value, comprising: administering SIRT3 oran agonist thereof to the subject.

Various embodiments provide for a method of selecting a treatment fordementia and/or neurological condition in a subject, comprising:analyzing a biological sample from the subject for PACAP level and/orSIRT3 level; and prescribing PACAP, SIRT3, agonists thereof, saltsthereof, or derivatives thereof, for the subject when the PACAP level isbelow a PACAP reference value and/or the SIRT3 level is below the SIRT3reference value.

Various embodiments provide for a composition, comprising: PACAP, SIRT3,an agonists of a receptor to PACAP, an agonist of a receptor to SIRT3,salts thereof, derivatives thereof, or pharmaceutical equivalentsthereof, or combinations thereof; and an pharmaceutically acceptablecarrier.

Other features and advantages of the invention will become apparent fromthe following detailed description, taken in conjunction with theaccompanying drawings, which illustrate, by way of example, variousfeatures of embodiments of the invention.

BRIEF DESCRIPTION OF THE FIGURES

Exemplary embodiments are illustrated in referenced figures. It isintended that the embodiments and figures disclosed herein are to beconsidered illustrative rather than restrictive.

FIGS. 1A-1E depict, in accordance with embodiments herein, PACAP levelsare inversely related to AD pathology. FIGS. 1A-1D. PACAP level in ADbrains were analyzed for correlation with amyloid plaque quantity(indicated by CERAD Plaque score). PACAP levels were inverselycorrelated with CERAD in the ENT (Pearson r=−0.6764, p<0.05, FIG. 1A)and SFG (Pearson r=−0.7088, p<0.05, FIG. 1C) but not in the MTG (FIG.1B) or PVC (FIG. 1D). PACAP level was quantified by ELISA and normalizedwith total protein (mg) of the brain tissue. FIG. 1E. PACAP level in CSFwas quantified and correlated with tau pathology (indicated by BraakStage). All CN cases were in Stage Four AD cases were in Stage III-IVwhile the other 5 AD cases in Stage V-VI. PACAP was lower in advancedBraak Stage V-VI than that in moderate Braak Stage III-IV (p<0.05).PACAP level was quantified by ELISA and normalized with CSF volume (ml).ENT=Entorhinal Cortex, MTG=Middle Temporal Lobe, SFG=Superior FrontalCortex, PVC=Primary Visual Cortex, AD=Alzheimer's disease (blue dot),CN=cognitively normal controls (black dot).

FIGS. 2A-2B depict, in accordance with embodiments herein, reduction inPACAP levels in CSF is specific to AD. PACAP levels were quantified byELISA as ng PACAP per ml undiluted CSF. FIG. 2A. PACAP was reduced in ADbut not in PDD or FTLD. FIG. 2B. PACAP is correlated with the Z score ofDRS. CN and AD cases were separated by the dotted line based on theirDRS score. AD=Alzheimer's disease, CN=cognitively normal controls,PDD=Parkinson Disease with Dementia, FTLD=Frontotemporal Lobe Dementia,DRS=Dementia Rating Scale-Revised (DRS-R), a global cognitiveassessment.

* indicates p<0.05 and **p<0.01.

FIGS. 3A-3D depict, in accordance with embodiments herein, comparisonsof PACAP abundance in selected cerebral regions among CN, MCI and AD.FIG. 3A: PACAP in CSF was expressed as ng PACAP per ml CSF. FIG. 3B:PACAP in SFG. FIG. 3C: PACAP in MTG. FIG. 3D: PACAP in PVC. In FIGS. 3B,3C, and 3D, PACAP was expressed as ng PACAP per mg protein from the samesample. Bars indicate standard errors. One way ANOVA with Post hoc Tukeytest. * indicates p<0.05, **p<0.01, ***p<0.001.

FIGS. 4A-4D depict, in accordance with embodiments herein, PACAP levelin selected cerebral regions correlate with region-specific cognitivetests.

FIGS. 4E-4F depict, in accordance with embodiments herein, PACAP in CSFinversely correlate with total amyloid plaque and total tangles. Solidfitted lines indicate significant Pearson correlations (p<0.05).

FIGS. 5A-5E depict, in accordance with embodiments herein, PAC1 receptorin selected cerebral regions and the PACAP-PAC1 receptor interaction.FIG. 5A. PAC1 receptor quantification in SFG, * indicate p<0.05, one wayANOVA with post hoc Tukey's tests. FIG. 5B. PAC1 receptor quantificationin MTG. FIG. 5C. PAC1 receptor quantification in MTG. FIG. 5D.Pharmacodynamic model fit of SFG PACAP and SFG PAC1 level to predict thecorrelation with Stroop Color-Word Interference z scores. The dashedline marks the point at significant correlation (p<0.05). FIG. 5E.Pharmacodynamic model fit of MTG PACAP and MTG PAC1 levels to predictthe correlation with AVLT scores. The dashed line marks the point atsignificant correlation (p<0.05).

DESCRIPTION OF THE INVENTION

All references cited herein are incorporated by reference in theirentirety as though fully set forth. Unless defined otherwise, technicaland scientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs. Hornyak, et al., Introduction to Nanoscience andNanotechnology, CRC Press (2008); Singleton et al., Dictionary ofMicrobiology and Molecular Biology 3rd ed., J. Wiley & Sons (New York,N.Y. 2001); March, Advanced Organic Chemistry Reactions, Mechanisms andStructure 7th ed., J. Wiley & Sons (New York, N.Y. 2013); and Sambrookand Russel, Molecular Cloning: A Laboratory Manual 4th ed., Cold SpringHarbor Laboratory Press (Cold Spring Harbor, N.Y. 2012), provide oneskilled in the art with a general guide to many of the terms used in thepresent application. One skilled in the art will recognize many methodsand materials similar or equivalent to those described herein, whichcould be used in the practice of the present invention. Indeed, thepresent invention is in no way limited to the methods and materialsdescribed.

As used herein, “PACAP” is an abbreviation of Pituitary AdenylateCyclase Activating Polypeptide.

As used herein, “AD” is an abbreviation of Alzheimer's disease.

As used herein, “SIRT” is an abbreviation of sirtuin.

As used herein, “analog” of a molecule such as a peptide refers to amolecule similar in function to either the entire molecule or to afragment thereof. Analogs typically differ from naturally occurringpeptides at one or a few positions, often by virtue of conservativesubstitutions. Analogs typically exhibit at least 80 or 90% sequenceidentity with natural peptides. Some analogs also include unnaturalamino acids or modifications of N or C terminal amino acids. Examples ofunnatural amino acids are, for example but not limited to; disubstitutedamino acids, N-alkyl amino acids, lactic acid, 4-hydroxyproline,γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine,O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine,5-hydroxylysine, σ-N-methylarginine. Fragments and analogs can bescreened for prophylactic or therapeutic efficacy in transgenic animalmodels.

As used herein, “conservative amino acid substitutions” result fromreplacing one amino acid with another having similar structural and/orchemical properties, such as the replacement of a leucine with anisoleucine or valine, an aspartate with a glutamate, or a threonine witha serine. Thus, a “conservative substitution” of a particular amino acidsequence refers to substitution of those amino acids that are notcritical for polypeptide activity or substitution of amino acids withother amino acids having similar properties (e.g., acidic, basic,positively or negatively charged, polar or non-polar, etc.) such thatthe substitution of even critical amino acids does not reduce theactivity of the peptide, (e.g., the ability of the peptide to penetratethe blood brain barrier (BBB)). Conservative substitution tablesproviding functionally similar amino acids are well known in the art.For example, the following six groups each contain amino acids that areconservative substitutions for one another: 1) Alanine (A), Serine (S),Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine(N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I),Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F),Tyrosine (Y), Tryptophan (W). In some embodiments, individualsubstitutions, deletions or additions that alter, add or delete a singleamino acid or a small percentage of amino acids can also be considered“conservative substitutions” if the change does not reduce the activityof the peptide. Insertions or deletions are typically in the range ofabout 1 to 5 amino acids. The choice of conservative amino acids may beselected based on the location of the amino acid to be substituted inthe peptide, for example if the amino acid is on the exterior of thepeptide and expose to solvents, or on the interior and not exposed tosolvents.

In alternative embodiments, one can select the amino acid which willsubstitute an existing amino acid based on the location of the existingamino acid, i.e., its exposure to solvents (i.e., if the amino acid isexposed to solvents or is present on the outer surface of the peptide orpolypeptide as compared to internally localized amino acids not exposedto solvents). Selection of such conservative amino acid substitutionsare well known in the art, for example as disclosed in Dordo et al, J.MoI Biol, 1999, 217, 721-739 and Taylor et al, J. Theor. Biol.119(1986); 205-218 and S. French and B. Robson, J. MoI. Evol.19(1983)171. Accordingly, one can select conservative amino acidsubstitutions suitable for amino acids on the exterior of a protein orpeptide (i.e., amino acids exposed to a solvent), for example, but notlimited to, the following substitutions can be used: substitution of Ywith F, T with S or K, P with A, E with D or Q, N with D or G, R with K,G with N or A, T with S or K, D with N or E, I with L or V, F with Y, Swith T or A, R with K, G with N or A, K with R, A with S, K or P.

In alternative embodiments, one can also select conservative amino acidsubstitutions encompassed suitable for amino acids on the interior of aprotein or peptide, for example one can use suitable conservativesubstitutions for amino acids is on the interior of a protein or peptide(i.e. the amino acids are not exposed to a solvent).

As used herein, “derivative” refers to peptides which have beenchemically modified, for example but not limited to by techniques suchas ubiquitination, labeling, pegylation (derivatization withpolyethylene glycol), lipidation, glycosylation, or addition of othermolecules. A molecule also a “derivative” of another molecule when itcontains additional chemical moieties not normally a part of themolecule. Such moieties can improve the molecule's solubility,absorption, biological half-life, etc. The moieties can alternativelydecrease the toxicity of the molecule, eliminate or attenuate anyundesirable side effect of the molecule, etc.

As further described herein, Pituitary Adenylate Cyclase ActivatingPolypeptide (PACAP) is a neurotrophin. The inventors studied the brainsof pathologically confirmed late onset AD patients and age matchedcognitively normal (CN) subjects to investigate the expression of PACAPmRNA (34 AD and 14 CN) and protein (12 AD and 11 CN). They found thatPACAP levels are reduced in multiple brain regions, including theentorhinal cortex (ENT), the middle temporal gyms (MTG), the superiorfrontal gyms (SFG) and the primary visual cortex (PVC). This reductionis inversely correlated with amyloid burden (CERAD plaque density) inthe ENT and SFG but not the PVC, a region spared in most cases of AD.PACAP expression is lower in the advanced Braak Stage (V-VI) than thatin the moderate stage (III-IV). PACAP level is correlated with DementiaRating Scale, a global cognitive assessment. Furthermore, PACAP level incerebrospinal fluid reflects its level in the brain and is reduced in ADbut not in Parkinson's disease dementia or Frontotemporal lobe dementia.The close inverse relationship between PACAP reduction and ADpathological markers suggests that down regulation of PACAP contributesto AD pathogenesis.

Further, AD is associated with a characteristic and progressive patternof reductions in regional cerebral metabolism as measured byflourodeoxyglucose positron emission tomography (FDG PET). Thesereductions begin years before the onset of cognitive symptoms and arecorrelated with clinical severity. Evidence supports the possibilitythat mitochondrial dysfunction is an initiating factor leading toapoptosis which is a common pathological mechanism forneurodegeneration. Insufficient energy metabolism due to complex Imalfunction may contribute to tau phosphorylation, a crucialpathological step in Alzheimer's disease. PACAP and Sirts targetmitochondria to improve its function. In addition, PACAP and Sirt 3 havea direct protective effect on neurons, likely targeting on intrinsicapoptotic pathway. The inventors found that PACAP and SIRT3 expressionis reduced in AD patients and the triple transgenic mouse model of AD.PACAP and SIRT3 protects against AP induced cell death by enhancingmitochondrial function. In one embodiment, PACAP and SIRT3, individuallyand/or collectively, can be used as a biomarker for AD. In anotherembodiment, PACAP and SIRT3, individually and/or collectively, may beused as a treatment for AD. In another embodiment, PACAP and/or SIRT3will improve mitochondria function. In another embodiment, PACAP and/orSIRT3 will rescue neurons from apoptosis.

We show that PACAP levels start to decline before the onset of AD, asearly as the MCI stage. This reduction in PACAP is region specific,targeting vulnerable regions of AD. These data support the possibilitythat the PACAP deficit is a risk factor for AD pathogenesis.Furthermore, the PACAP deficit in selected cerebral regions may predictregion-specific cognitive function deterioration. We have found thestrongest correlation between PACAP levels and cognitive performance inSFG and MTG, two regions heavily involved in AD pathology, and both ofwhich represent cognitive abilities that are affected early in thecourse of AD. The CSF PACAP level inversely correlates with the totalquantities of amyloid plaques and tangle plaques. PACAP specificreceptor PAC1 showed a transient upregulation in the frontal lobe of MCIsubjects but not in AD patients, suggesting a potential compensatorymechanism in MCI. AD patients may lose this compensatory capability asthe disease progresses.

Using ligand-receptor pharmacodymamic model, we estimated that anapproximate 1:12:1 ratio of PACAP:PAC1 in SFG predicts the StroopColor-Word Interference task performance. In the temporal lobe however,PACAP has to be excessive (˜5 fold) to reach a significant correlationwith cognitive performance. This is consistent with our data that showslower levels of PAC1 receptor in MTG compared to SFG. An alternativeexplanation is that a large proportion of measured PACAP in MTG isintracellularly retained and unavailable for paracrine secretion, as ourmeasurement did not discern multiple compartments in tissues; or thereis another subtype of PAC1 that has weak ligand-receptor interaction inthis region. Taken together, the action target of PACAP is predominantlylocalized in the frontal lobe. This MTG-SFG pathway is consistent withthe proposed neurodegenerative network most affected in AD.

The PACAP level in SFG is higher than that of MTG regardless incontrols, MCIs or AD patients. But the PACAP deficit in AD is moresevere in MTG (˜60% reduction) than in SFG (45% reduction).Interestingly, the total Aβ in SFG was increased 10 fold in AD comparedto the control, whereas Aβ in MTG was increased 100 fold in AD. Thisregional difference in Aβ deposition is consistent with the inverserelationship between PACAP and amyloid load, as shown herein.

Although the human studies on PACAP are in the preliminary stage, animalstudies have shown beneficial effects of PACAP on cognition and memory.The earliest clue of PACAP's effects on memory came from amnesiac (amn)gene mutant flies. Amn gene encodes a homolog of vertebral PACAP. PACAPis also essential for associative memory in Lymnaea stagnalis, aprototype model for studying the molecular mechanism of simpleassociative learning. Most importantly, intranasal administration ofPACAP enhances Novel object recognition in SAMP8 mice and APP transgenicmice. The memory enhancing effect of PACAP may be mediated by multiplemolecular mechanisms, including enhancing inward currents through NMDAchannel and AMPA channel, inhibiting HCN channel or A-type K channel.

Various embodiments of the present invention are based, at least inpart, on these findings.

Diagnosis of Dementia and Neurological Conditions

In various embodiments, the present invention provides a method ofdiagnosing dementia and/or a neurological condition in an individual byobtaining a sample from the individual, preparing the sample to beassayed by a serological test to determine the presence or absence ofPACAP expression levels, and diagnosing dementia and/or a neurologicalcondition based on the presence of low PACAP expression levels relativeto those levels found in a healthy subject. In various embodiments, themethod further comprises preparing the sample to be assayed by aserological test to determine the presence or absence of SIRT3expression levels, and diagnosing dementia and/or a neurologicalcondition based on the presence of low PACAP expression levels and lowSIRT3 expression levels relative to those levels found in a healthysubject

Various embodiments of the present invention provides for a method fordetermining whether a subject has dementia, comprising: obtaining abiological sample from a subject; determining the PACAP expression levelin the biological sample, and diagnosing the subject as having dementiaif low PACAP expression levels relative to a reference value is found inthe biological sample. In various embodiments, the method furthercomprises determining the SIRT3 expression level in the biologicalsample, and diagnosing the subject as having dementia if low PACAPexpression levels and low SIRT3 expression levels relative to areference value are found in the biological sample

Various embodiments of the present invention provides for a method fordetermining whether a subject has a neurological condition, comprising:obtaining a biological sample from a subject; determining the PACAPexpression level in the biological sample, and diagnosing the subject ashaving the neurological condition if low PACAP expression levelsrelative to a reference value is found in the biological sample. Invarious embodiments, the method further comprises determining the SIRT3expression level in the biological sample, and diagnosing the subject ashaving the neurological condition if low PACAP expression levels and lowSIRT3 expression levels relative to a reference value are found in thebiological sample

Various embodiments provide for a method of diagnosing dementiacomprising obtaining a biological sample from a subject desiring adiagnosis regarding dementia; assaying the biological sample todetermine PACAP expression level; and determining the subject hasdementia if the PACAP expression level is lower than a reference value,or determining the subject does not have dementia if the PACAPexpression level is not lower than the reference value. In variousembodiments, the method further comprises assaying the biological sampleto determine SIRT3 expression level; and determining the subject hasdementia if the PACAP expression level and the SIRT3 expression levelare lower than their reference values, or determining the subject doesnot have dementia if the PACAP expression level and the SIRT3 expressionlevel are not lower than their reference values.

In various embodiments, the present invention provides for a method ofdiagnosing dementia and/or a neurological condition in an individual byobtaining a sample from the individual, preparing the sample to beassayed by a serological test to determine the presence or absence ofSIRT3 expression levels, and diagnosing dementia and/or a neurologicalcondition based on the presence of low SIRT3 expression levels relativeto those levels found in a healthy subject.

Various embodiments of the present invention provides for a method fordetermining whether a subject has dementia, comprising: obtaining abiological sample from a subject; determining the SIRT3 expression levelin the biological sample, and diagnosing the subject as having dementiaif low SIRT3 expression levels relative to a reference value is found inthe biological sample.

Various embodiments of the present invention provides for a method fordetermining whether a subject has a neurological condition, comprising:obtaining a biological sample from a subject; determining the SIRT3expression level in the biological sample, and diagnosing the subject ashaving the neurological condition if low SIRT3 expression levelsrelative to a reference value is found in the biological sample.

Various embodiments provide for a method of diagnosing dementiacomprising obtaining a biological sample from a subject desiring adiagnosis regarding dementia; assaying the biological sample todetermine SIRT3 expression level; and determining the subject hasdementia if the SIRT3 expression level is lower than a reference value,or determining the subject does not have dementia if the SIRT3expression level is not lower than the reference value.

Various embodiments provide for a method of diagnosing a neurologicalcondition comprising obtaining a biological sample from a subjectdesiring a diagnosis regarding dementia; assaying the biological sampleto determine SIRT3 expression level; and determining the subject has theneurological condition if the SIRT3 expression level is lower than areference value, or determining the subject does not have theneurological condition if the SIRT3 expression level is not lower thanthe reference value.

In various embodiments, the invention provides for a system fordiagnosing dementia and/or neurological condition in a subject,comprising: a biological sample from the subject; an antibody thatspecifically binds to PACAP and/or an antibody that specifically bindsto SIRT3; and a machine to detect the binding of the antibody PACAPand/or the antibody to SIRT3.

In various embodiments, the invention provides for a composition fordiagnosing dementia and/or neurological condition in a subject,comprising: a biological sample obtained from the subject; and anantibody that specifically binds to PACAP and/or an antibody thatspecifically binds to SIRT3.

In various embodiments, the invention provides for a system fordiagnosing dementia and/or neurological condition in a subject,comprising: an antibody-PACAP complex and/or an antibody-SRIT3 complexin a biological sample obtained from the subject; and a machine todetect the antibody-PACAP complex and/or an antibody-SIRT3 complex.

In various embodiments, the invention provides for a composition fordiagnosing dementia and/or neurological condition in a subject,comprising: an antibody-PACAP complex and/or an antibody-SIRT3 complexin a biological sample obtained from the subject.

In various embodiments, PACAP is the full length form (PACAP-38). Invarious embodiments, PACAP is the short form (PACAP-27). In otherembodiments, PACAP is both the full length form and the short form.

In various embodiments, the dementia is Alzheimer's disease (AD). Invarious embodiments, the individual or subject is human.

Prognosis of Dementia and Neurological Conditions

In various embodiments, the present invention provides a method ofprognosing dementia and/or a neurological condition in an individual byobtaining a sample from the individual, preparing the sample to beassayed by a serological test to determine the presence or absence ofPACAP expression levels, and prognosing a severe form of dementia and/ora neurological condition based on the presence of low PACAP expressionlevels relative to those levels found in a healthy subject. In variousembodiments, the method further comprises preparing the sample to beassayed by a serological test to determine the presence or absence ofSIRT3 expression levels, and prognosing a severe form of dementia and/ora neurological condition based on the presence of low PACAP expressionlevels and low SIRT3 expression levels relative to those levels found ina healthy subject. In another embodiment, the dementia is Alzheimer'sdisease (AD). In another embodiment, the individual is human.

In another embodiment, the present invention provides a method ofprognosing dementia and/or a neurological condition in an individual byobtaining a sample from the individual, preparing the sample to beassayed by a serological test to determine the presence or absence ofSIRT3 expression levels, and prognosing a severe form of dementia and/ora neurological condition based on the presence of low SIRT3 expressionlevels relative to those levels found in a healthy subject. In anotherembodiment, the dementia is Alzheimer's disease (AD). In anotherembodiment, the individual is human.

Various embodiments of the present invention provides for a method forprognosing dementia, comprising: obtaining a biological sample from asubject; determining the PACAP expression level in the biologicalsample, and prognosing the subject as having a severe form of dementiaif low PACAP expression levels relative to a reference value is found inthe biological sample. In various embodiments, the method furthercomprises determining the SIRT3 expression level in the biologicalsample, and prognosing the subject as having a severe form of dementiaif low PACAP expression levels and low SIRT3 expression levels relativetheir reference values are found in the biological sample.

Various embodiments of the present invention provides for a method forprognosing a neurological condition, comprising: obtaining a biologicalsample from a subject; determining the PACAP expression level in thebiological sample, and prognosing the subject as having a severe form ofthe neurological condition if low PACAP expression levels relative to areference value is found in the biological sample. In variousembodiments, the method further comprises determining the SIRT3expression level in the biological sample, and prognosing the subject ashaving a severe form of the neurological condition if low PACAPexpression levels and low SIRT3 expression levels relative to theirreference values are found in the biological sample.

In various embodiments, PACAP is the full length form (PACAP-38). Invarious embodiments, PACAP is the short form (PACAP-27). In otherembodiments, PACAP is both the full length form and the short form.

Distinguishing Between of Alzheimer's Disease and Parkinson's Disease

Various embodiments of the present invention provides for a method fordistinguishing whether a subject has Alzheimer's disease or Parkinson'sdisease, comprising: obtaining a biological sample from a subject;determining the PACAP expression level in the biological sample, anddiagnosing the subject as having Alzheimer's disease if low PACAPexpression levels relative to a reference value is found in thebiological sample. In various embodiments, the method further comprisesdetermining the SIRT3 expression level in the biological sample, anddiagnosing the subject as having Alzheimer's disease if low PACAPexpression levels and low SIRT3 expression levels relative to areference value are found in the biological sample.

Various embodiments provide for a method of distinguishing betweenAlzheimer's disease and Parkinson's disease comprising obtaining abiological sample from a subject desiring a distinction betweenAlzheimer's disease and Parkinson's disease; assaying the biologicalsample to determine PACAP expression level; and determining the subjecthas Alzheimer's disease if the PACAP expression level is lower than areference value, or determining the subject does not have Alzheimer'sdisease if the PACAP expression level is not lower than the referencevalue. In various embodiments, the method further comprises assaying thebiological sample to determine SIRT3 expression level; and determiningthe subject has Alzheimer's disease if the PACAP expression level andthe SIRT3 expression level are lower than their reference values, ordetermining the subject does not have Alzheimer's disease if the PACAPexpression level and the SIRT3 expression level are not lower than theirreference values.

In various embodiments, the individual or subject is human.

In various embodiments, PACAP is the full length form (PACAP-38). Invarious embodiments, PACAP is the short form (PACAP-27). In otherembodiments, PACAP is both the full length form and the short form.

Treatment of Dementia and Neurological Conditions

In another embodiment, the present invention provides a method oftreating dementia and/or a neurological condition in an individual byproviding a composition comprising SIRT3, or a salt, analog, derivative,pharmaceutical equivalent thereof, and administering a therapeuticallyeffective dosage of the composition to the individual. In anotherembodiment, the composition is administered to the individualintravenously. In another embodiment, the composition is administeredintranasally. In another embodiment, the dementia is Alzheimer's disease(AD). In another embodiment, the individual is human.

In another embodiment, the present invention provides a method oftreating dementia and/or a neurological condition by providing acomposition comprising one or more agonists of receptors to PACAP orSIRT3, and administering a therapeutically effective dosage of thecomposition to the individual. In another embodiment, the dementia isAlzheimer's disease.

In various embodiments, the present invention provides a method oftreating dementia and/or neurological condition in an individual byadministering a therapeutically effective dosage of a compositioncomprising one or more neurotrophins to the individual.

In various embodiments, the present invention provides a method oftreating dementia and/or neurological condition, comprisingadministering a therapeutically effective dosage of a compositioncomprising one or more neurotrophins to a subject who has beendetermined to have dementia and/or the neurological condition by amethod of the present invention.

In various embodiments, the present invention provides a method oftreating dementia and/or neurological condition, comprising providing acomposition comprising one or more neurotrophins; and administering atherapeutically effective dosage of the composition to a subject who hasbeen determined to have dementia and/or the neurological condition by amethod of the present invention.

In various embodiments, the present invention provides for a method fortreating dementia and/or neurological condition in a subject,comprising: analyzing a biological sample from the subject for a PACAPlevel; and administering a neurotrophin or an agonist of a neurotrophinto the subject when the PACAP level is below a reference value to treatdementia and/or neurological condition. In various embodiments, themethod further comprises analyzing the biological sample for a SIRT3level and a neurotrophin or an agonist of a neurotrophin is administeredto the subject when the PACAP level is below a PACAP reference value andthe SIRT3 level is below a SIRT3 reference value to treat dementiaand/or neurological condition.

In various embodiments, the present invention provides for a method fortreating dementia and/or neurological condition in a subject,comprising: obtaining the results of an analysis of a PACAP level in asubject; and administering a neurotrophin or an agonist of aneurotrophin to the subject when the PACAP level is below a referencevalue. In various embodiments, the method further comprises obtainingthe results of an analysis a SIRT3 level and the neurotrophin or anagonist of the neurotrophin is administered to the subject when thePACAP level is below a PACAP reference value and the SIRT3 level isbelow a SIRT3 reference value to treat dementia and/or neurologicalcondition.

In various embodiments, the present invention provide for a method fortreating dementia and/or neurological condition in a subject who hasbeen determined to have a PACAP level below a reference value,comprising: administering a neurotrophin or an agonist thereof to thesubject. In various embodiments, the subject has also been determined tohave a SIRT3 level below a SIRT3 reference value.

In various embodiments, the present invention provides for a method fortreating dementia and/or neurological condition in a subject,comprising: analyzing a biological sample from the subject for a SIRT3level; and administering a SIRT3 or an agonist thereof to the subjectwhen the SIRT3 level is below a reference value to treat dementia and/orneurological condition.

In various embodiments, the present invention provides for a method fortreating dementia and/or neurological condition in a subject,comprising: obtaining the results of an analysis of a SIRT3 level in asubject; and administering SIRT3 or an agonist thereof to the subjectwhen the SIRT3 level is below a reference value.

In various embodiments, the present invention provide for a method fortreating dementia and/or neurological condition in a subject who hasbeen determined to have a SIRT3 level below a reference value,comprising: administering SIRT3 or an agonist thereof to the subject.

In various embodiments, the present invention provides for a method ofselecting a treatment for dementia and/or neurological condition in asubject, comprising: analyzing a biological sample from the subject forPACAP level and/or SIRT3 level; and prescribing PACAP, SIRT3, agoniststhereof, salts thereof, derivatives thereof, for the subject when thePACAP level is below a PACAP reference value and/or the SIRT3 level isbelow the SIRT3 reference value.

In various embodiments, the analysis or determination of PACAP and/orSIRT3 levels are done via the methods described herein.

In various embodiments, the neurotrophin includes PACAP, or a salt,derivative, or pharmaceutical equivalent thereof. In another embodiment,the composition is administered intravenously. In various embodiments,PACAP is the full length form (PACAP-38). In various embodiments, PACAPis the short form (PACAP-27). In other embodiments, PACAP is both thefull length form and the short form.

In another embodiment, the individual is treated by decreasing amyloidplaque in the individual.

In another embodiment, the dementia is Alzheimer's disease.

Biological Samples

Biological samples used in accordance with various embodiments of thepresent invention can be mammalian body fluids, sera such as blood(including whole blood as well as its plasma and serum), CSF (spinalfluid), urine, sweat, saliva, tears, pulmonary secretions, breastaspirate, prostate fluid, seminal fluid, stool, cervical scraping,cysts, amniotic fluid, intraocular fluid, mucous, moisture in breath,animal tissue, cell lysates, tumor tissue, hair, skin, buccal scrapings,nails, bone marrow, cartilage, prions, bone powder, ear wax, etc. oreven from external or archived sources such as tumor samples (i.e.,fresh, frozen or paraffin-embedded). Samples, such as body fluids orsera, obtained during the course of clinical trials may be advantageousfor, although samples obtained directly from living subjects underalternate conditions or for other purposes may be readily used as well.In various embodiments, the biological sample is cerebrospinal fluid(CSF). In various embodiments, the biological sample is plasma. Invarious embodiments, the biological sample is serum.

Reference Values PACAP Reference Values

In various embodiments, the reference value can be the median or meanADCYAP1 (the PACAP gene) expression level from a population of subjectswith without dementia or the neurological condition.

The nucleic acid samples used to compute a reference value are takenfrom at least 1, 2, 5, 10, 20, 30, 40, 50, 100, or 200 differentorganisms of that species. According to certain aspects of theinvention, nucleic acid “derived from” genomic DNA, as used in themethods of the invention, e.g., in hybridization experiments todetermine ADCYAP1 expression can be fragments of genomic nucleic acidgenerated by restriction enzyme digestion and/or ligation to othernucleic acid, and/or amplification products of genomic nucleic acids,pre-messenger RNA (pre-mRNA), or post-messenger RNA (the mature form ofmRNA), amplification products of pre- or post-mRNA, or genomic DNAfragments grown up in cloning vectors generated, e.g., by “shotgun”cloning methods. In certain embodiments, genomic nucleic acid samplesare digested with restriction enzymes.

In various embodiments, the reference value can be the median or meanPACAP protein expression level from a population of subjects withwithout dementia or the neurological condition.

The protein samples used to compute a reference value are taken from atleast 1, 2, 5, 10, 20, 30, 40, 50, 100, or 200 different organisms ofthat species.

SIRT3 Reference Value

In various embodiments, the reference value can be the median or meanSIRT3 expression level from a population of subjects with withoutdementia or the neurological condition.

The nucleic acid samples used to compute a reference value are takenfrom at least 1, 2, 5, 10, 20, 30, 40, 50, 100, or 200 differentorganisms of that species. According to certain aspects of theinvention, nucleic acid “derived from” genomic DNA, as used in themethods of the invention, e.g., in hybridization experiments todetermine SIRT3 expression can be fragments of genomic nucleic acidgenerated by restriction enzyme digestion and/or ligation to othernucleic acid, and/or amplification products of genomic nucleic acids,pre-messenger RNA (pre-mRNA), or post-messenger RNA (the mature form ofmRNA), amplification products of pre- or post-mRNA, or genomic DNAfragments grown up in cloning vectors generated, e.g., by “shotgun”cloning methods. In certain embodiments, genomic nucleic acid samplesare digested with restriction enzymes.

In various embodiments, the reference value can be the median or meanSIRT3 protein expression level from a population of subjects withwithout dementia or the neurological condition.

The protein samples used to compute a reference value are taken from atleast 1, 2, 5, 10, 20, 30, 40, 50, 100, or 200 different organisms ofthat species.

Determining Expression Levels and Assays

In various embodiments, determining the PACAP expression level orassaying the biological sample to determine PACAP expression levelcomprises detecting PACAP (ADCYAP1) mRNA with a polynucleotide capableof hybridizing with PACAP (ADCYAP1) mRNA under stringent hybridizationconditions.

In various embodiments, determining the PACAP expression level orassaying the biological sample to determine PACAP expression level(ADCYAP1 expression level) comprises using DNA sequencing, mRNAexpression assay, RT-PCR, real-time PCR, or a combination thereof.

In various embodiments, determining the PACAP expression level orassaying the biological sample to determine PACAP expression levelcomprises contacting the biological sample with an anti-PACAP antibodyand detecting whether binding occurs or the amount of binding occurringbetween PACAP protein and the anti-PACAP antibody. In variousembodiments, wherein the anti-PACAP antibody binds to an epitope that ispresent on PACAP.

In various embodiments, detecting whether binding occurs between PACAPprotein and the anti-PACAP antibody comprises using flow cytometry.

In various embodiments, determining the PACAP expression level orassaying the biological sample to determine PACAP expression levelcomprises detecting a PACAP protein with an antibody capable ofspecifically binding to a PACAP protein.

In various embodiments, determining the PACAP expression level orassaying the biological sample to determine PACAP expression levelcomprises using enzyme-linked immunosorbent assay (ELISA), including butnot limited to indirect ELISA, sandwich ELISA, competitive ELISA,multiple and portable ELISA.

In various embodiments, determining the PACAP expression level orassaying the biological sample to determine PACAP expression levelcomprises using immunohistochemistry, flow cytometry, fluorescence insitu hybridization (FISH), radioimmuno assays, and affinitypurification.

In various embodiments, determining the SIRT3 expression level orassaying the biological sample to determine SIRT3 expression levelcomprises detecting SIRT3 mRNA with a polynucleotide capable ofhybridizing with SIRT3 mRNA under stringent hybridization conditions.

In various embodiments, determining the SIRT3 expression level orassaying the biological sample to determine SIRT3 expression levelcomprises using DNA sequencing, mRNA expression assay, RT-PCR, real-timePCR, or a combination thereof.

In various embodiments, determining the SIRT3 expression level orassaying the biological sample to determine SIRT3 expression levelcomprises contacting the biological sample with an anti-SIRT3 antibodyand detecting whether binding occurs or the amount of binding occurringbetween SIRT3 protein and the anti-SIRT3 antibody. In variousembodiments, wherein the anti-SIRT3 antibody binds to an epitope that ispresent on SIRT3. In various embodiments, detecting whether bindingoccurs between SIRT3 protein and the anti-SIRT3 antibody comprises usingflow cytometry.

In various embodiments, determining the SIRT3 expression level orassaying the biological sample to determine SIRT3 expression levelcomprises detecting a SIRT3 protein with an antibody capable ofspecifically binding to a SIRT3 protein.

In various embodiments, determining the SIRT3 expression level orassaying the biological sample to determine SIRT3 expression levelcomprises using enzyme-linked immunosorbent assay (ELISA), including butnot limited to indirect ELISA, sandwich ELISA, competitive ELISA,multiple and portable ELISA.

In various embodiments, determining the SIRT3 expression level orassaying the biological sample to determine SIRT3 expression levelcomprises using immunohistochemistry, flow cytometry, fluorescence insitu hybridization (FISH), radioimmuno assays, and affinitypurification.

In one embodiment, the present invention provides a compositioncomprising PACAP, or a salt, derivative, or pharmaceutical equivalentthereof, and an acceptable carrier. In another embodiment, the presentinvention provides a composition comprising SIRT3, or a salt,derivative, or pharmaceutical equivalent thereof, and an acceptablecarrier. In another embodiment, the present invention provides acomposition comprising PACAP and SIRT3, and an acceptable carrier. Inanother embodiment, the present invention provides a compositioncomprising one or more agonists of receptors of PACAP or SIRT3, and anacceptable carrier.

In various embodiments, the present invention provides pharmaceuticalcompositions including a pharmaceutically acceptable excipient alongwith a therapeutically effective amount of SIRT3, PACAP, and/or one ormore agonists of receptors to SIRT3 or PACAP. “Pharmaceuticallyacceptable excipient” means an excipient that is useful in preparing apharmaceutical composition that is generally safe, non-toxic, anddesirable, and includes excipients that are acceptable for veterinaryuse as well as for human pharmaceutical use. Such excipients may besolid, liquid, semisolid, or, in the case of an aerosol composition,gaseous.

In various embodiments, the pharmaceutical compositions according to theinvention may be formulated for delivery via any route ofadministration. “Route of administration” may refer to anyadministration pathway known in the art, including but not limited toaerosol, nasal, oral, transmucosal, transdermal or parenteral.“Parenteral” refers to a route of administration that is generallyassociated with injection, including intraorbital, infusion,intraarterial, intracapsular, intracardiac, intradermal, intramuscular,intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal,intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous,transmucosal, or transtracheal. Via the parenteral route, thecompositions may be in the form of solutions or suspensions for infusionor for injection, or as lyophilized powders.

The pharmaceutical compositions according to the invention can alsocontain any pharmaceutically acceptable carrier. “Pharmaceuticallyacceptable carrier”, or “acceptable carrier”, as used herein refers to apharmaceutically acceptable material, composition, or vehicle that isinvolved in carrying or transporting a compound of interest from onetissue, organ, or portion of the body to another tissue, organ, orportion of the body. For example, the carrier may be a liquid or solidfiller, diluent, excipient, solvent, or encapsulating material, or acombination thereof. Each component of the carrier must be“pharmaceutically acceptable” in that it must be compatible with theother ingredients of the formulation. It must also be suitable for usein contact with any tissues or organs with which it may come in contact,meaning that it must not carry a risk of toxicity, irritation, allergicresponse, immunogenicity, or any other complication that excessivelyoutweighs its therapeutic benefits.

The pharmaceutical compositions according to the invention can also beencapsulated, tableted or prepared in an emulsion or syrup for oraladministration. Pharmaceutically acceptable solid or liquid carriers maybe added to enhance or stabilize the composition, or to facilitatepreparation of the composition. Liquid carriers include syrup, peanutoil, olive oil, glycerin, saline, alcohols and water. Solid carriersinclude starch, lactose, calcium sulfate, dihydrate, terra alba,magnesium stearate or stearic acid, talc, pectin, acacia, agar orgelatin. The carrier may also include a sustained release material suchas glyceryl monostearate or glyceryl distearate, alone or with a wax.

The pharmaceutical preparations are made following the conventionaltechniques of pharmacy involving milling, mixing, granulation, andcompressing, when necessary, for tablet forms; or milling, mixing andfilling for hard gelatin capsule forms. When a liquid carrier is used,the preparation will be in the form of a syrup, elixir, emulsion or anaqueous or non-aqueous suspension. Such a liquid formulation may beadministered directly p.o. or filled into a soft gelatin capsule.

The pharmaceutical compositions according to the invention may bedelivered in a therapeutically effective amount. The precisetherapeutically effective amount is that amount of the composition thatwill yield the most effective results in terms of efficacy of treatmentin a given subject. This amount will vary depending upon a variety offactors, including but not limited to the characteristics of thetherapeutic compound (including activity, pharmacokinetics,pharmacodynamics, and bioavailability), the physiological condition ofthe subject (including age, sex, disease type and stage, generalphysical condition, responsiveness to a given dosage, and type ofmedication), the nature of the pharmaceutically acceptable carrier orcarriers in the formulation, and the route of administration. Oneskilled in the clinical and pharmacological arts will be able todetermine a therapeutically effective amount through routineexperimentation, for instance, by monitoring a subject's response toadministration of a compound and adjusting the dosage accordingly. Foradditional guidance, see Remington: The Science and Practice of Pharmacy(Gennaro ed. 22^(nd) edition, Williams & Wilkins PA, USA) (2012).

Typical dosages of a therapeutically effective dosage of SIRT3 or PACAP,or agonists of receptors to SIRT3 or PACAP can be in the rangesrecommended by the manufacturer where known therapeutic compounds areused, and also as indicated to the skilled artisan by the in vitroresponses or responses in animal models. Such dosages typically can bereduced by up to about one order of magnitude in concentration or amountwithout losing the relevant biological activity. Thus, the actual dosagewill depend upon the judgment of the physician, the condition of thepatient, and the effectiveness of the therapeutic method based, forexample, on the in vitro responsiveness of the relevant primary culturedcells or histocultured tissue sample, such as biopsied malignant tumors,or the responses observed in the appropriate animal models, aspreviously described.

The present invention is also directed to a kit to diagnose, prognose,or treat Alzheimer's disease or a neurological disorder. The kit isuseful for practicing the inventive method of diagnosing or prognosingthe disorder by determining the presence of one or more biomarkers,including expression levels of PACAP and/or SIRT3. The kit is anassemblage of materials or components, including at least one of theinventive compositions.

The exact nature of the components configured in the inventive kitdepends on its intended purpose. For example, some embodiments areconfigured for the purpose of treating Alzheimer's disease. In oneembodiment, the kit is configured particularly for the purpose oftreating mammalian subjects. In another embodiment, the kit isconfigured particularly for the purpose of treating human subjects. Infurther embodiments, the kit is configured for veterinary applications,treating subjects such as, but not limited to, farm animals, domesticanimals, and laboratory animals.

Instructions for use may be included in the kit. “Instructions for use”typically include a tangible expression describing the technique to beemployed in using the components of the kit to effect a desired outcome.Optionally, the kit also contains other useful components, such as,diluents, buffers, pharmaceutically acceptable carriers, syringes,catheters, applicators, pipetting or measuring tools, bandagingmaterials or other useful paraphernalia as will be readily recognized bythose of skill in the art.

The materials or components assembled in the kit can be provided to thepractitioner stored in any convenient and suitable ways that preservetheir operability and utility. For example the components can be indissolved, dehydrated, or lyophilized form; they can be provided atroom, refrigerated or frozen temperatures. The components are typicallycontained in suitable packaging material(s). As employed herein, thephrase “packaging material” refers to one or more physical structuresused to house the contents of the kit, such as inventive compositionsand the like. The packaging material is constructed by well knownmethods, preferably to provide a sterile, contaminant-free environment.As used herein, the term “package” refers to a suitable solid matrix ormaterial such as glass, plastic, paper, foil, and the like, capable ofholding the individual kit components. The packaging material generallyhas an external label which indicates the contents and/or purpose of thekit and/or its components.

The various methods and techniques described above provide a number ofways to carry out the invention. Of course, it is to be understood thatnot necessarily all objectives or advantages described may be achievedin accordance with any particular embodiment described herein. Thus, forexample, those skilled in the art will recognize that the methods can beperformed in a manner that achieves or optimizes one advantage or groupof advantages as taught herein without necessarily achieving otherobjectives or advantages as may be taught or suggested herein. A varietyof advantageous and disadvantageous alternatives are mentioned herein.It is to be understood that some preferred embodiments specificallyinclude one, another, or several advantageous features, while othersspecifically exclude one, another, or several disadvantageous features,while still others specifically mitigate a present disadvantageousfeature by inclusion of one, another, or several advantageous features.

Furthermore, the skilled artisan will recognize the applicability ofvarious features from different embodiments. Similarly, the variouselements, features and steps discussed above, as well as other knownequivalents for each such element, feature or step, can be mixed andmatched by one of ordinary skill in this art to perform methods inaccordance with principles described herein. Among the various elements,features, and steps some will be specifically included and othersspecifically excluded in diverse embodiments.

Although the invention has been disclosed in the context of certainembodiments and examples, it will be understood by those skilled in theart that the embodiments of the invention extend beyond the specificallydisclosed embodiments to other alternative embodiments and/or uses andmodifications and equivalents thereof.

Many variations and alternative elements have been disclosed inembodiments of the present invention. Still further variations andalternate elements will be apparent to one of skill in the art. Amongthese variations, without limitation, are the selection of constituentmodules for the inventive compositions, and the diseases and otherclinical conditions that may be diagnosed, prognosed or treatedtherewith. Various embodiments of the invention can specifically includeor exclude any of these variations or elements.

In some embodiments, the numbers expressing quantities of ingredients,properties such as concentration, reaction conditions, and so forth,used to describe and claim certain embodiments of the invention are tobe understood as being modified in some instances by the term “about.”Accordingly, in some embodiments, the numerical parameters set forth inthe written description and attached claims are approximations that canvary depending upon the desired properties sought to be obtained by aparticular embodiment. In some embodiments, the numerical parametersshould be construed in light of the number of reported significantdigits and by applying ordinary rounding techniques. Notwithstandingthat the numerical ranges and parameters setting forth the broad scopeof some embodiments of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspracticable. The numerical values presented in some embodiments of theinvention may contain certain errors necessarily resulting from thestandard deviation found in their respective testing measurements.

In some embodiments, the terms “a” and “an” and “the” and similarreferences used in the context of describing a particular embodiment ofthe invention (especially in the context of certain of the followingclaims) can be construed to cover both the singular and the plural. Therecitation of ranges of values herein is merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g. “such as”) provided with respectto certain embodiments herein is intended merely to better illuminatethe invention and does not pose a limitation on the scope of theinvention otherwise claimed. No language in the specification should beconstrued as indicating any non-claimed element essential to thepractice of the invention.

Groupings of alternative elements or embodiments of the inventiondisclosed herein are not to be construed as limitations. Each groupmember can be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. One ormore members of a group can be included in, or deleted from, a group forreasons of convenience and/or patentability. When any such inclusion ordeletion occurs, the specification is herein deemed to contain the groupas modified thus fulfilling the written description of all Markushgroups used in the appended claims.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations on those preferred embodiments will become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Itis contemplated that skilled artisans can employ such variations asappropriate, and the invention can be practiced otherwise thanspecifically described herein. Accordingly, many embodiments of thisinvention include all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

Furthermore, numerous references have been made to patents and printedpublications throughout this specification. Each of the above citedreferences and printed publications are herein individually incorporatedby reference in their entirety.

In closing, it is to be understood that the embodiments of the inventiondisclosed herein are illustrative of the principles of the presentinvention. Other modifications that can be employed can be within thescope of the invention. Thus, by way of example, but not of limitation,alternative configurations of the present invention can be utilized inaccordance with the teachings herein. Accordingly, embodiments of thepresent invention are not limited to that precisely as shown anddescribed.

EXAMPLES

The following examples are provided to better illustrate the claimedinvention and are not to be interpreted as limiting the scope of theinvention. To the extent that specific materials are mentioned, it ismerely for purposes of illustration and is not intended to limit theinvention. One skilled in the art may develop equivalent means orreactants without the exercise of inventive capacity and withoutdeparting from the scope of the invention.

Example 1 Methods

Postmortem human brains were obtained from the Banner Sun HealthResearch Institute Brain and Body Donation Program (BBDP). Theoperations of the BBDP have been approved by the Western InstitutionalReview Board. Frozen brain tissues were obtained from patients with aclinical and pathological diagnosis of late onset AD and fromage-matched cognitive normal subjects (CN). Brain donors all underwentextensive longitudinal clinical and neuropsychological assessmentantemortem. All AD cases were selected as being “intermediate” or “high”probability for AD according to NIA-Reagan criteria (National Instituteon Aging-Alzheimer's Association criteria). CN subjects did not meet thecriteria for AD or dementia. The AD cases and controls did not differsignificantly in their age at death, gender, or educational level.Cortical Aβ neuritic plaque density (CERAD evaluation) and Braak tanglestage were determined by a neuropathologist (T.G.B). Postmortemcisternal CSF samples from the same cohort were obtained.

Protein sample solution and CSF (AD=12 and CN=11) were quantified forPACAP expression using standard ELISA kit (Cat #MBS160511, MyBiosourceInc. San Diego, Calif.) according to the manufacture protocol. Briefly,samples were loaded and incubated with biotin-labeled PACAP antibody at370 C for 60 minutes. The plated was washed with the washing buffer forfive times, followed by incubation with chromogen solution at 370 C for10 minutes. The reaction was stopped by adding stop solution. The finalresults were read at OB 450 nm. The ELISA result of PACAP levels in thebrain were compared to the western blot result. The PACAP level measuredwas in the middle range of the standard curve and was correlated nicelywith the western blot results (CN: Pearson's r=0.9093, P<0.01; AD:Pearson's r=0.8243, P<0.01).

Another set of postmortem brain samples (AD=34, CN=14) were used fortranscriptome study. Briefly, brain sections were stained with acombination of Thioflavin-S (Sigma-Aldrich, Dallas, Tex.) and 1% neutralred (Fisher Scientific, Chicago, Ill.). Pyramidal neurons wereidentified and laser captured onto Arcturus CapSure Macro LCM Caps andextracted according to the manufacturer's protocol. Total RNA wasisolated from the neuronal cell lysate with the Arcturus PicoPure RNAIsolation Kit with DNase I treatment using Qiagen's RNase-free DNase Set(Valencia, Calif.). Isolated total RNA from each sample of ˜500 neuronswas double-round amplified, cleaned, and biotin labeled withAffymetrix's GeneChip per the manufacturer's protocol. Amplified andlabeled cRNA was quantitated on a spectrophotometer and run on a 1%Tris-acetate-EDTA (TAE) gel to check for an evenly distributed range oftranscript sizes 8.

T-tests were used to compare values of two groups. For comparing valuesacross multiple groups, a one way ANOVA with post-hoc Tukey's test wasused. Pearson's correlation assay was applied for correlation analyses.All results were reported as mean±standard error. Statisticalsignificance was set at P<0.05.

PACAP is Reduced in Multiple Areas of Human AD Brain.

Neurons were laser captured and micro-dissected from multiple brainregions of AD patients and CN subjects. ADCYAP1 (the PACAP gene)expression was significantly reduced in AD (Table1.1). Overall, theinventors identified significantly decreased neuronal expression ofADCYAP1 in the Middle Temporal Gyms (MTG), Superior Frontal Gyms (SFG),and Primary Visual Cortex (PVC). To validate thisneurotranscriptome-based screening, they selected a different cohort of12 cases of AD postmortem brain samples and 11 CN cases. They used ELISAto quantify PACAP protein expression. The PACAP protein levels werereduced in AD in all three regions and entorhinal cortex (ENT) (Table1.2).

TABLE 1.1 Neurotranscriptome of ADCYAP1 gene expression Gene Brainregion Fold change P value ADCYAP1 ENT −3.702532581 0.127300685 ADCYAP1MTG −4.039581566 0.005626227 ADCYAP1 SFG −8.911001119 0.008381943ADCYAP1 PVC −5.904469845 0.00228059 ADCYAP1 is the PACAP gene. ENT =Entorhinal Cortex, MTG = Middle Temporal Lobe, SFG = Superior FrontalCortex, PVC = Primary Visual Cortex

TABLE 1.2 ELISA quantification of PACAP CN AD PACAP PACAP (Mean ± (Mean± SEM, ×10⁻² ng/mg SEM, ×10⁻² ng/mg protein) N protein) N P value ENT2.13 ± 0.25 11 1.41 ± 0.19 12 0.035 MTG 1.24 ± 0.11 8 0.73 ± 0.05 110.007 SFG 6.35 ± 0.33 8 3.63 ± 0.22 10 0.001 PVC 5.98 ± 0.35 7 3.86 ±0.45 11 0.004

PACAP levels were normalized with total protein (mg) of the braintissue. Data were presented as Mean+standard error. ENT=EntorhinalCortex, MTG=Middle Temporal Lobe, SFG=Superior Frontal Cortex,PVC=Primary Visual Cortex, AD=Alzheimer's disease, CN=cognitively normalcontrols.

PACAP Reduction is Associated with Pathological Hallmarks of AD

Amyloid plaques and neurofibrillary tangle are the two pathologicalhallmarks of AD. PACAP protein levels were inversely correlated withCERAD amyloid plaque score in the ENT and SFG but not in the MTG or PVC(FIGS. 1A-1D), suggesting that lower PACAP level is associated with amore pronounced AP deposition. In terms of neurofibrillary tangles, theAD cases had Braak stages ranging from IV to VI, whereas the CN samplesranged from III-IV. PACAP levels were reduced in Braak stage V-VI (allAD cases) than in stage III-IV (FIG. 1E). It's noteworthy that in thesame Braak stage AD cases had lower PACAP levels than CN cases;suggesting PACAP might be a more sensitive biomarker than tau pathology.

PACAP Levels in CSF Reflex its Levels in Brain

In AD cases, the PACAP level in CSF was reduced as compared with CN(1.83±0.11 ng/ml in AD, N=9, vs. 2.10±0.04 ng/ml in CN, N=7, p<0.01,FIG. 2A). In contrast, PACAP levels in Parkinson Disease with Dementia(PDD) (2.11±0.08 ng per ml CSF, N=8) and in Frontotemporal Lobe Dementia(FTLD) (2.01±0.07 ng per ml CSF, N=8) was comparable to that of CN (FIG.2A). Furthermore, the PACAP level in CSF was strongly correlated withthe Mattis Dementia Rating Scale-Revised (DRS-R), a measure of globalcognitive functioning (Pearson's r=0.8197, p=0.0001, FIG. 2B herein).

Generally

In the four brain areas examined, both PACAP mRNA transcription andprotein expression are significantly reduced in AD brains compared tomatched CN controls. PACAP levels in the CSF were also significantlydecreased in AD versus controls. This supports that the reducedneurotrophic effects from PACAP is an important factor contributing toAlzheimer's pathology.

The CERAD amyloid plaque burden is inversely correlated with PACAPexpression in the ENT and SFG, but not the MTG or PVC. This is notsurprising because amyloid deposition usually spares the PVC unless inthe case of posterior cortical atrophy. This concurs with the animalstudy showing that PACAP may inhibit the amyloidogenic processing andfacilitate amyloid clearance. The progression of neurofibrillary tangleis defined by Braak stage that follows the pathway from the entorhinalcortex to the temporal lobe and then to the distal neocortex. PACAPlevels at stage III-IV (limbic) was relatively higher than those at theadvanced stage V-VI (isocortical). Thus, PACAP is associated with bothpathological hallmarks of AD.

PACAP as a neurotrophin is abundant in the brain. PACAP levels in theCSF can be a good surrogate for diagnosis and monitoring the diseaseprogression since CSF is more readily available than brain biopsy. PACAPlevels in the CSF are reduced in AD but not in PDD or FTLD, suggestingit is more specific to AD. This is consistent with the closerelationship between PACAP and pathological markers of AD.

In summary, PACAP, an effective neurotrophin, is reduced in thepathonogmonic cortical regions of AD. PACAP levels inversely correlatewith AD pathology. In addition to its role as an effective biomarker,therapeutic effects have been shown in animal models of AD.

Example 2 Methods

Postmortem human cerebral cortex and cerebrospinal fluid (CSF) wereobtained from the Arizona Alzheimer's Disease Center and the Banner SunHealth Research Institute Brain and Body Donation Program (BBDP),including 16 AD, 9 MCI and 10 CN subjects. All AD cases were selected asbeing “intermediate” or “high” probability for AD according toNIA-Reagan criteria (National Institute on Aging-Alzheimer's Associationcriteria) (Consensus recommendations for the postmortem diagnosis ofAlzheimer's disease. The National Institute on Aging, and ReaganInstitute Working Group on Diagnostic Criteria for the NeuropathologicalAssessment of Alzheimer's Disease 1997). In addition, they were free ofother neurodegenerative disorders such as vascular dementia, Parkinson'sdisease, dementia with Lewy bodies, frontotemporal dementia, hippocampalsclerosis, progressive supranuclear palsy, dementia lacking distinctivehistology, multiple system atrophy, motor neuron disease with dementiaand corticobasal degeneration. MCI was diagnosed by consensus as asyndrome of cognitive impairment beyond age-adjusted norms that is notsevere enough to impair daily function or fulfill clinical criteria fordementia. To ensure MCI is an early stage of AD, we excluded 3 cases ofMCI that didn't have AD pathology. All patients underwent longitudinalclinical and neuropsychological assessment antemortem. Since PACAPlevels may change with age, only the final antemortem assessment scorewithin one year prior to death, if available, was included for PACAPcorrelation analysis. The raw scores from Mattis Dementia Rating Scale(DRS), Stroop Color-Word Interference trial, the total words learnedover trials (new learning) score, Auditory Verbal Learning Test totallearning (AVLT-TL) and Judgment of Line Orientation (JLO) were convertedto age- and education-corrected z scores.

We measured samples from parenchymal cortical homogenate includingsuperior frontal gyms (SFG), middle temporal gyms (MTG), primary visualcortex (PVC), and from cerebrospinal fluid (CSF) samples. Proteinsamples and CSF were quantified for PACAP and PAC1 receptor usingstandard ELISA kit (Cat #MBS160511 and MBS042704, MyBiosource Inc. SanDiego, Calif.) according to the manufacture protocol. Briefly, proteinsamples were loaded and incubated with biotin-labeled PACAP antibody orPAC1 receptor antibody at 37° C. for 60 minutes. The plate was washedwith the washing buffer for five times, followed by incubation withchromogen solution at 37° C. for 10 minutes. The reaction was stopped byadding stop solution. The final results were read at OB 450 nm. Inparallel, the protein quantity was determined with Pierce BCA proteinassay kit (Cat #23227, Thermo Scientific, Rockford, Ill.). To eliminatethe confounding factor of potential neurodegeneration, we normalized thePACAP level to the protein level in the brain tissue. Therefore, PACAPin cortical tissues was expressed as ng per mg total protein, whereasPACAP in CSF was expressed as ng/ml CSF.

We used ANOVA with post hoc Tukey pairwise comparisons and Pearsoncorrelations, setting p<0.05 as the level of significance. All resultsare presented as mean±S.D in Table 2 and mean±S.E. in figures. Wehypothesized the PACAP-PAC1 interaction would produce a net biologicaleffect, which correlated with specific cognitive function. The ligand(L)-receptor (R) interaction pharmacodynamic model obeys the chemicalkinetic principle. In this case, L represents PACAP and R representsPAC1 receptor. We assumed that the biological activity (cognitiveperformance in our case) is determined by the interaction of n mole ofPACAP with each mole of PAC1 receptor. Therefore,n[L]+[R]=[LnR]-->Biological activity. At equilibrium, Kd=[LnR]/[L]^(n)[R], where Kd is the disassociation factor. Therefore, we hypothesizedthat the z score of cognitive performance is linearly proportional to[L]^(n) [R]. We did a series of linear correlation analyses between theproduct of [PACAP]^(n)×[PAC1] and cognitive performance z scores.

Results

Patient demographic data and final antemortem cognitive scores aresummarized in the Table 2.

TABLE 2 Patient demographic and cognitive data Mild Cognitive CognitiveAlzheimer Normal Impairment Disease (CN) (MCI) (AD)^(#) Total casenumber 10 9 16 Gender (M/F) 8/2 4/5 7/9 Age, Mean (SD), years 86.3(5.7)  87.2 (4.6) 81.7 (8.9) Most recent antemortem cognitive taskperformance^(#) DRS z-score, Mean 0.95 (0.50) −0.85 (0.81)*** −2.31(0.65)*** (SP) Stroop Words and 0.38 (1.05) −0.83 (1.17) −1.87 (1.11)***Color Interference z score, Mean (SD) AVLT-TL z score, 0.34 (0.96) −1.15(0.89)* −2.58 (1.73)*** Mean (SD) JLO z score, Mean (SD) 1.33 (1.05)0.93 (0.84) −1.03 (1.53)** ^(#)Cognitive testing done within a year ofdeath. DRS: Dementia Rating Scale-II, Stroop Color-Word InterferenceTrial, AVLT-TL: Auditory Verbal Learning Test total learning, JLO:Judgment of Line Orientation. All scores converted to z-scores based onage- and education-corrected norms. *p < 0.05, **P < 0.01, ***p < 0.001,compared to CN, one way ANOVA with post hoc Tukey analysis

The average age was similar among the three groups. PACAP levels in CSFwere reduced in AD patients (1.61±0.06 ng/ml, n=16, p<0.01) compared toCN subjects (2.08±0.08 ng/ml, n=10). Noteworthy, PACAP in MCI patients(1.80±0.07 ng/ml, n=9, p<0.05) was also significantly reduced, albeit toa lesser extent (FIG. 3A). This progressive reduction of PACAP from CNto MCI and to AD was also apparent in SFG and MTG. PACAP in SFG (FIG.3B) was reduced in MCI [(3.77±0.26)×10⁻² ng/mg, n=9, p<0.01] and ADpatients [(2.85±0.19)×10⁻² ng/mg, n=16, p<0.001] compared to CN subjects[(5.87±0.31)×10⁻² ng/mg, n=10]. PACAP in MTG (FIG. 3C) in MCI[(0.95±0.16)×10⁻² ng/mg, n=8, p<0.001] and in AD patients[(0.75±0.06)×10⁻² ng/mg, n=16, p<0.01] was reduced compared to that ofCN subjects [(1.95±0.31)×10⁻² ng/mg, n=10]. In PVC, PACAP (FIG. 3D) wasreduced in AD patients [(4.62±0.39)×10⁻² ng/mg, n=16, p<0.05] but not inMCI patients [(5.31±0.36)×10⁻² ng/mg, n=7, p=0.18], compared to CNsubjects [(6.93±0.71)×10′ ng/mg, n=8]. Therefore, PACAP reduction isevident at MCI stage that precedes the onset of AD in the expected brainregions.

Since cognitive decline can occur in multiple cognitive domains, weanalyzed the relationship between PACAP levels and cognitiveperformance. PACAP levels in CSF were correlated with DRS z-scores(Pearson r=0.503, p<0.05, FIG. 4A), suggesting the reduction ofaggregated PACAP levels in CNS is correlated with poorer globalcognitive function (DRS). Furthermore, PACAP levels in SFG werecorrelated with Stroop Color-Word Interference z-scores (Pearsonr=0.584, p<0.01, FIG. 4B), a task that is sensitive to frontal lobefunction. The PACAP levels in the MTG correlated with the AVLT-TL(Pearson r=0.329, p<0.05, FIG. 4C), a task that is sensitive to temporallobe function. By contrast, PACAP levels in PVC did not correlate withJLO (p=0.14, FIG. 4D), a visuospatial task sensitive to occipitoparietalfunctioning. In addition, the CSF PACAP level inversely correlates withthe total amyloid plaque number (r=−0.4805, p<0.01, FIG. 4E) and totaltangles numbers (r=−0.5464, p=0.01, FIG. 4F).

Since PACAP exert its physiological function by binding to PAC1receptors in the brain, we measured the quantity of PAC1 receptors inSFG, MTG and PVC. As PAC1 receptors are located on the cell membrane,they are not detectable in CSF. In SFG, the PAC1 level in the AD group(199±22 ng/mg, n=16) was not different from that of controls (213±25ng/mg, n=10, FIG. 5A). Interestingly, PAC1 level in MCI (312±25 ng/mg,n=9, p<0.05) was higher than that of controls (FIG. 5A). However, thisPAC1 receptor upregulation in MCI was not observed in MTG or PVC. PAC1in MTG was 58±14 in CN (n=9), 67±10.4 in MCI (n=8) and 61±7.9 in AD(n=16, p=0.47, FIG. 5B). PAC1 in PVC was 34±8.2 in CN (n=9), 48±9.5 inMCI (n=8) and 44±4.2 in AD (n=16, p=0.51, FIG. 5C). The relativeabundance of PAC1 receptor was SFG>>MTG>PVC, and was not affected by thedisease stages, i.e. MCI or AD.

According to the pharmacodynamic model, the interaction of ligand andreceptor determines the biological activity. We hypothesized that theproduct of [PACAP]^(n)×[PAC1R] would predict the cognitive function.Based on the algorithms described in detail in methods, a series ofPearons R and associated correlation P value are calculated by varyingligand numbers in the range of zero to ten. The relationship betweenPearsons R (or P value) and ligand number (n) was fit into a polynomialrelationship. In the SFG, 2 PCACP for each PAC 1 receptor was requiredto reach a P value of less than 0.05 (FIG. 5D). This suggests a ratio ofPACAP:PAC1 receptor at 2:1 best predicts the Stroop Word and Colorinteraction cognitive function in SFG. In MTG, more PACAP, at least 5,was required to bind to the PAC1 receptor to reach significantcorrelation with AVLT-TL. In PVC, P value nadir (P=0.16) did not reachsignificance regardless of the ligand number, suggesting the PACAP-PAC1interaction in visual cortex did not predict JLO. Thus, theligand-receptor interaction predicts regional cognitive function best inSFG, less so in MTG, and failed in PVC.

Various embodiments of the invention are described above in the DetailedDescription. While these descriptions directly describe the aboveembodiments, it is understood that those skilled in the art may conceivemodifications and/or variations to the specific embodiments shown anddescribed herein. Any such modifications or variations that fall withinthe purview of this description are intended to be included therein aswell. Unless specifically noted, it is the intention of the inventorsthat the words and phrases in the specification and claims be given theordinary and accustomed meanings to those of ordinary skill in theapplicable art(s).

The foregoing description of various embodiments of the invention knownto the applicant at this time of filing the application has beenpresented and is intended for the purposes of illustration anddescription. The present description is not intended to be exhaustivenor limit the invention to the precise form disclosed and manymodifications and variations are possible in the light of the aboveteachings. The embodiments described serve to explain the principles ofthe invention and its practical application and to enable others skilledin the art to utilize the invention in various embodiments and withvarious modifications as are suited to the particular use contemplated.Therefore, it is intended that the invention not be limited to theparticular embodiments disclosed for carrying out the invention.

While particular embodiments of the present invention have been shownand described, it will be obvious to those skilled in the art that,based upon the teachings herein, changes and modifications may be madewithout departing from this invention and its broader aspects and,therefore, the appended claims are to encompass within their scope allsuch changes and modifications as are within the true spirit and scopeof this invention. It will be understood by those within the art that,in general, terms used herein are generally intended as “open” terms(e.g., the term “including” should be interpreted as “including but notlimited to,” the term “having” should be interpreted as “having atleast,” the term “includes” should be interpreted as “includes but isnot limited to,” etc.).

1. A method of detecting PACAP and SIRT3 protein expression in a humansubject, the method comprising the steps of: obtaining a sample from thehuman subject; detecting an expression level of PACAP protein in thesample; and detecting an expression level of SIRT3 protein in thesample.
 2. The method of claim 1, wherein the sample comprises at leastone of a brain tissue or cerebrospinal fluid.
 3. The method of claim 1,wherein the expression level of PACAP protein in the sample is detectedusing at least one of ELISA, Western blot, and flow cytometry.
 4. Themethod of claim 1, wherein the human subject is suspected of having lateonset Alzheimer's disease (LOAD).
 5. The method of claim 1, wherein thedetecting the expression level of PACAP protein and detecting the levelof SIRT3 protein in the sample comprises using antibody-based assays. 6.The method of claim 1 and further comprising comparing the detectedexpression level of PACAP protein in the subject's sample to a referencelevel of PACAP protein.
 7. The method of claim 6 and further comprisingadministering a composition comprising PACAP to the subject if thesubject's expression level of PACAP is below that of the reference levelof PACAP protein.
 8. The method of claim 1, wherein the human subjectsuspected of having at least one of late onset Alzheimer's disease(LOAD), Parkinson's disease with Dementia (PDD), and Frontotemporal LobeDementia (FTLD).
 9. The method of claim 8 and further comprisingadministering a composition comprising PACAP to the human subject. 10.The method of claim 8 and further comprising comparing the detectedexpression level of PACAP protein in the subject's sample to a referencelevel of PACAP protein.
 11. The method of claim 12, wherein humansubjects with LOAD exhibit reduced levels of PACAP expression comparedto human subjects with PDD and FTLD.
 12. A method of detecting PACAPprotein expression in a human subject exhibiting symptoms of late onsetAlzheimer's disease (LOAD), the method comprising the steps of:obtaining a sample from the human subject exhibiting symptoms of LOAD,wherein the sample comprises at least one of a brain tissue orcerebrospinal fluid; and detecting, using an antibody-based assay, anexpression level of PACAP protein in the sample.
 13. The method of claim12, wherein the expression level of PACAP protein in the sample isdetected using at least one of ELISA, Western blot, and flow cytometry.14. The method of claim 12 and further comprising comparing the detectedexpression level of PACAP protein in the subject's sample to a referencelevel of PACAP protein.
 15. The method of claim 12 and furthercomprising administering a composition comprising PACAP to the subjectif the subject's expression level of PACAP is below that of thereference level of PACAP protein.
 16. A method of detecting PACAPprotein expression in a human subject exhibiting symptoms of late onsetAlzheimer's disease (LOAD): obtaining a cerebrospinal fluid sample fromthe human subject; preparing the sample to be assayed by a serologicaltest to determine a PACAP expression level; detecting, using anantibody-based assay, the PACAP protein expression level in thecerebrospinal fluid sample from the human subject.
 17. The method ofclaim 16 and further comprising comparing the detected expression levelof PACAP protein in the subject's sample to a reference level of PACAPprotein.
 18. The method of claim 16, wherein the expression level ofPACAP protein in the sample is detected using at least one of ELISA,Western blot, and flow cytometry.
 19. The method of claim 16 and furthercomprising administering a composition comprising PACAP to the humansubject.
 20. The method of claim 16 and further comprising measuring anexpression level of SIRT3 protein in the sample.